Chimeric mice with highly humanized livers have multiple potential applications in research and preclinical drug development both in the academic and commercial settings [1]. The applications include studies of human infectious diseases (hepatitis B and B, malaria etc.), drug metabolism and pharmacokinetics, drug-drug interactions, toxicology and gene therapy among others. In the current human chimeric liver models up to 90% of hepatocytes are of human origin and display all the typical functions of mature human cells. Our company uses immune deficient fumarylacetoacetate hydrolase (Fah) knockout mice[2], bearing additional mutations in Rag2 and the interleukin common gamma chain receptor (Il2rg) designated as FRGN to generate such chimeric animals. Human hepatocytes from commercial vendors are transplanted into the animals and over time the human hepatocytes replace the mouse cells generating chimeric animals with livers that constitute 90% human cells. Currently, hepatocytes are the only human cell type present in FRGN chimeric livers and this represents a limitation for some applications of the model. Specifically, hematopoietic cell types are known to contribute significantly to liver biology, including pathobiology. Inflammation is present in many settings, particularly in liver infections, but also chemical injuries. Furthermore, the liver macrophages (Kupffer cells) are important players in liver toxicology. All of the hematopoietic cells in the current generation of chimeric FRGN mice are of murine origin and they are completely deficient in T- and B-cells rendering the in sufficient for the afore mentioned studies. Chimeric mice bearing significant percentages of all human hematopoietic cells can be readily generated by transplantation of human cord blood into immune deficient mice, including Rag2/Il2rg/Nod mice. In this application we propose to optimize a protocol that permits the efficient generation of double- chimeric FRGN mice, with high levels of human hematopoiesis as well as high percentages of liver repopulation. Aim 1: To optimize a transplant protocol in FRGN mice which reproducibly yields >70% human chimerism in both the liver and blood. 1a) Different time points for transplantation and different protocols or conditioning by irradiation will be compared using a single human hepatocyte donor. 2a) the two best protocols will be applied to 3 additional hepatocyte donors validate and further optimize the procedure. PUBLIC HEALTH RELEVANCE: Drug metabolism is phenotypically specific and can vary significantly between individuals of the same species and the variance between individuals can be attributed to genetic differences in the expression and activity of liver enzymes responsible for the metabolism of the drugs as well as significant contributions by the immune system in response to the metabolites produced by the liver. Yecuris Corporation has developed an in vivo genetic selection system (the FRG KO mouse) that permits extensive humanization of murine liver by transplanted human hepatocytes. With the addition of a humanized hematopoietic system in conjunction with the humanized liver, the new dual chimeric model could revolutionize development and screening of therapeutics in addition to providing models for studying hepatic infectious diseases.